Our long range goal is to understand the intracellular signaling pathways that are activated by antigen and cytokine receptors on lymphocytes and how they regulate outcomes such as cell growth, differentiation and effector functions. This proposal focuses on the adapter protein ShcA (referred to here onwards as Shc) and how it regulates signaling events downstream of the pre-TCR during thymic T cell development. Our previous studies using conditional knockout mice deficient in Shc protein expression showed a requirement for Shc in progression through the pre-TCR- dependent ? selection checkpoint. We also identified a key for tyrosine phosphorylation of Shc at this step, using mice inducibly expressing a dominant negative mutant of Shc. Our recent work suggests a dominant role for Shc in leading to ERK activation during ? selection, and that Shc specifically regulates proliferation and differentiation events downstream of the pre-TCR (but not allelic exclusion or cell survival). In this proposal, we will attempt to dissect the specific molecular mechanisms by which Shc regulates these events during thymic development. In Aim 1, we will use a combination of in vivo and ex vivo approaches to address the requirement and sufficiency of Ras and ERK signals downstream of Shc. We will also take unbiased and focused approaches to probe the role of Shc binding partners for their role during ? selection. In Aim 2, we will follow up on our recent unexpected observation that Shc plays a role in migration of DN thymocytes via the chemokine receptor CXCR4, and suggest a crosstalk between pre- TCR and CXCR4. Specifically, we will address the role of CXCR4 in regulating the ??selection checkpoint using mouse models. We will also address the molecular mechanism by which Shc regulates CXCR4 signaling in thymocytes, and whether Shc may be one point of crosstalk between the pre-TCR and CXCR4. The results from these studies should provide a better molecular understanding of events regulating the ? selection checkpoint and the role of adapter proteins. Since CXCR4 is also a coreceptor for HIV-1 entry, these studies may also have implications for pediatric AIDS. PUBLIC HEALTH RELEVANCE: Understanding molecular events involved in the development of T cells in the thymus is a fundamentally important question. The focus of this proposal will be to address the molecular mechanisms by the adapter protein Shc regulates T cell development in vivo. This has important implications for understanding the generation of competent T cells that can participate in immune responses. Secondly, the chemokine receptor CXCR4 is a coreceptor for HIV-1 entry into cells. Our preliminary studies suggesting a role for Shc in signaling via CXCR4 in thymocytes and the studies proposed here could provide important insights into chemokine-dependent regulation of ??selection, with implications for pediatric AIDS. Thus, we believe the outcome of these studies could have high degree of significance from a basic science perspective as well as human health.